TBK1 at the Crossroads of Inflammation and Energy Homeostasis in Adipose Tissue.

The noncanonical IKK family member TANK-binding kinase 1 (TBK1) is activated by pro-inflammatory cytokines, but its role in controlling metabolism remains unclear. Here, we report that the kinase uniquely controls energy metabolism. Tbk1 expression is increased in adipocytes of HFD-fed mice. Adipocyte-specific TBK1 knockout (ATKO) attenuates HFD-induced obesity by increasing energy expenditure; further studies show that TBK1 directly inhibits AMPK to repress respiration and increase energy storage. Conversely, activation of AMPK under catabolic conditions can increase TBK1 activity through phosphorylation, mediated by AMPK's downstream target ULK1. Surprisingly, ATKO also exaggerates adipose tissue inflammation and insulin resistance. TBK1 suppresses inflammation by phosphorylating and inducing the degradation of the IKK kinase NIK, thus attenuating NF-κB activity. Moreover, TBK1 mediates the negative impact of AMPK activity on NF-κB activation. These data implicate a unique role for TBK1 in mediating bidirectional crosstalk between energy sensing and inflammatory signaling pathways in both over- and undernutrition.

Design and synthesis of potent carboxylic acid DGAT1 inhibitors with high cell permeability.

A series of potent carboxylic acid DGAT1 inhibitors with high permeability were developed from a virtual screening hit. Strategies were employed to reduce Pgp substrate recognition and increase passive permeability, resulting in the discovery of a series showing good inhibition of cellular triglyceride synthesis. The mutagenic potential of prospective metabolites was evaluated in the Ames assay, and one aniline was shown to be devoid of mutagenicity.

PSNCBAM-1, a novel allosteric antagonist at cannabinoid CB1 receptors with hypophagic effects in rats.

BACKGROUND AND PURPOSE: Rimonabant (Acomplia, SR141716A), a cannabinoid CB1 receptor inverse agonist, has recently been approved for the treatment of obesity. There are, however, concerns regarding its side effect profile. Developing a CB1 antagonist with a different pharmacological mechanism may lead to a safer alternative. To this end we have screened a proprietary small molecule library and have discovered a novel class of allosteric antagonist at CB1 receptors. Herein, we have characterized an optimized prototypical molecule, PSNCBAM-1, and its hypophagic effects in vivo. EXPERIMENTAL APPROACH: A CB1 yeast reporter assay was used as a primary screen. PSNCBAM-1 was additionally characterized in [35S]-GTPgammaS, cAMP and radioligand binding assays. An acute rat feeding model was used to evaluate its effects on food intake and body weight in vivo. KEY RESULTS: In CB1 receptor yeast reporter assays, PSNCBAM-1 blocked the effects induced by agonists such as CP55,940, WIN55212-2, anandamide (AEA) or 2-arachidonoyl glycerol (2-AG). The antagonist characteristics of PSNCBAM-1 were confirmed in [35S]-GTPgammaS binding and cAMP assays and was shown to be non-competitive by Schild analyses. PSNCBAM-1 did not affect CB2 receptors. In radioligand binding assays, PSNCBAM-1 increased the binding of [3H]CP55,940 despite its antagonist effects. In an acute rat feeding model, PSNCBAM-1 decreased food intake and body weight. CONCLUSIONS AND IMPLICATIONS: PSNCBAM-1 exerted its effects through selective allosteric modulation of the CB1 receptor. The acute effects on food intake and body weight induced in rats provide a first report of in vivo activity for an allosteric CB1 receptor antagonist.

Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors.

Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.

Inhibition of virus-encoded thymidine kinase suppresses herpes simplex virus replication in vitro and in vivo.

Both herpes simplex virus type 1 (HSV-1) and HSV-2 encode a thymidine kinase enzyme which differs from cellular thymidine kinase in substrate specificity. Viral thymidine kinase enables the virus to replicate in cells that lack cellular thymidine kinase, namely those of the sensory neurons where the virus establishes, and periodically reactivates from, a latent state. Thymidine kinase-dependent HSV replication following viral reactivation at the site of latency is thought to precede the emergence of virus at mucosal surfaces. The ability to inhibit such an essential viral enzyme would potentially prevent HSV from replicating within neuronal tissue, and thus stop the recurrent disease cycle. Ro 32-2313 was designed as a selective and competitive inhibitor of HSV thymidine kinase and in vitro studies have confirmed this mechanism of action. In vivo evaluation of a soluble prodrug of Ro 32-2313, Ro 32-4397, was undertaken in murine models where pathogenesis was dependent upon viral replication in neuronal tissue. It was shown that in vivo administration of Ro 32-4397 (i) significantly reduced the viral titre detected in isolated dorsal root ganglia; (ii) prevented HSV-2-induced lethality in a systemic infection model; and (iii) reduced zosteriform lesion development in a model of dermal infection. Administration of Ro 32-4397 produced dose-related changes in viral pathogenicity towards those of the phenotype of a thymidine kinase-deficient virus. Overall, the study confirmed that thymidine kinase inhibitors can suppress the replication of HSV in vivo, and suggest that such inhibitors may reduce reactivation of the virus from latency if used prophylactically in recurrent HSV infection.

SAR, pharmacokinetics, safety, and efficacy of glucokinase activating 2-(4-sulfonylphenyl)-N-thiazol-2-ylacetamides: discovery of PSN-GK1.

Allosteric activators of the glucose-sensing enzyme glucokinase (GK) are currently attracting much interest as potential antidiabetic therapies because they can achieve powerful blood glucose lowering through actions in multiple organs. Here, the optimization of a weakly active high-throughput screening hit to (2 R)-2-(4-cyclopropanesulfonylphenyl)- N-(5-fluorothiazol-2-yl)-3-(tetrahydropyran-4-yl)propionamide (PSN-GK1), a potent GK activator with an improved pharmacokinetic and safety profile, is described. Following oral administration, this compound elicited robust glucose lowering in rats.

Glucokinase activator PSN-GK1 displays enhanced antihyperglycaemic and insulinotropic actions.

AIMS/HYPOTHESIS: We evaluated the insulinotropic and antihyperglycaemic actions of glucokinase activators (GKAs), especially through acute and subchronic studies in rodent diabetes models with (2R)-2-(4-cyclopropanesulphonylphenyl)-N-(5-fluorothiazol-2-yl)-3-(tetrahydropyran-4-yl)propionamide (PSN-GK1), a novel and potent GKA. MATERIALS AND METHODS: The action of PSN-GK1 on or in the following were investigated: (1) on human liver glucokinase, insulin secretion from MIN6 cells and 2-deoxy-D: -[(3)H]glucose (2-DG) uptake into rat hepatocytes; and (2) in Zucker diabetic fatty rats and in non-diabetic C57Bl/6, diabetic db/db and ob/ob mice. RESULTS: At 5 mmol/l glucose, PSN-GK1 activated glucokinase (4.3-fold, median effective concentration [EC(50)] 130 nmol/l), increased MIN6 insulin secretion (26-fold, EC(50) 267 nmol/l) and 2-DG hepatocytic uptake (threefold, EC(50) 1 micromol/l); at higher glucose concentrations, EC(50)s and fold-effectiveness were both lower. In C57Bl/6 mice, PSN-GK1 reduced blood glucose at 1 and 10 mg/kg (by mouth), but insulin was increased significantly at only the higher dose. In hyperinsulinaemic 10-mmol/l glucose clamps, PSN-GK1 increased 2-DG incorporation into liver glycogen sixfold, directly demonstrating liver effects. PSN-GK1 improved glycaemic profiles in db/db mice and Zucker diabetic fatty rats, diabetic animal models in which GKA efficacy has not previously been described, without causing hypoglycaemia. In ob/ob mice, it dose-dependently reduced excursions in OGTTs. Moreover, after subchronic administration, no tachyphylaxis was evident and glycaemia was improved without alterations to lipid levels, liver weight, glycogen content or body weight. CONCLUSIONS/INTERPRETATION: PSN-GK1 was potently antihyperglycaemic through its effects on insulin release and hepatic glucose metabolism. It is one of the most potent GKAs described in the literature and is active in diabetic animal models where GKAs have not been reported to show efficacy to date. Ongoing human trials are investigating the potential of this novel therapeutic approach.

Production of host shutoff-defective mutants of herpes simplex virus type 1 by inactivation of the UL13 gene.

Two mutants of HSV-1(SC16) carrying disrupted UL13 genes have been generated independently by recombination of wild-type genomic DNA with a plasmid-cloned copy of the UL13 gene containing multiple stop codons. The two mutants were shown to be deficient in UL13 gene expression by Western blotting of infected cells. A revertant virus, in which UL13 expression was restored to a near-normal level, was generated by recombination of one of the UL13-negative mutants with a plasmid carrying the wild-type UL13 gene. The replication of the two UL13-negative viruses in cell culture was somewhat reduced compared to their wild-type parent, and the viruses were unable to produce shutoff of host protein synthesis. The replication of the revertant virus was intermediate between that of the UL13-negative and wild-type viruses, as was its ability to produce host shutoff. Cells infected with the UL13-negative mutants were shown to contain much lower levels than normal of the UL41 gene product, which is known to be required for virion host shutoff. However, there was no significant difference between levels of the UL41 gene product in wild-type and mutant virions. The UL13-negative viruses exhibited different patterns of protein phosphorylation from wild-type virus when infected cells were metabolically labeled with [32P]-orthophosphate and when lysates of infected cells and of virions were subjected to in vitro phosphorylation. However, the UL41 gene product could still be phosphorylated in lysates of UL13-negative virions. We conclude that the UL13 gene is necessary to produce the virion host shutoff effect, but it seems unlikely that the role of UL13 is simply to activate the UL41 gene product by phosphorylation.

Herpes simplex virus type 1 gene UL13 encodes a phosphoprotein that is a component of the virion.

The UL13 open reading frame of herpes simplex virus type 1 (HSV-1) has been expressed in insect cells by a recombinant baculovirus and in Escherichia coli. In the latter case, the UL13 gene was fused to the gene for glutathione S-transferase (GST) to allow high-level expression of an 80-kDa GST-UL13 fusion protein. Antibody raised against the fusion protein reacted specifically with the 55-kDa UL13 gene product expressed by the recombinant baculovirus. This antibody also recognized a late phosphoprotein in HSV-1-infected cell lysates and a component of purified HSV-1 virions, both with the same electrophoretic mobility as the baculovirus-expressed protein. The virion component was efficiently phosphorylated in vitro by a virion-associated protein kinase. Using the same antibody, the probable homolog of the UL13 gene product was identified in HSV-2-infected cells and purified virions.

Antiviral activity of 5'-PAA and 5'-PFA phosphate esters of 2'-deoxyuridines.

The 5'-PAA and 5'-PFA phosphate esters of 5-bromo-2'-deoxyuridine (BUdR) were synthesized and their antiherpes virus activity was evaluated in vitro. Both compounds showed activity of the same order as the parent nucleoside, BUdR, against HSV-2 but were 4 to 12 times less potent against HSV-1. The 5'-PAA phosphate ester of BUdR (Ro 21-9875) was also active against varicella-zoster virus (VZV) and human cytomegalovirus (HCMV). The 5'-PAA phosphate ester of 5-bromovinyl-2'-deoxyuridine (BVdU) was also synthesized and showed good antiviral activity against HSV-1 only (ID50 = 1.3 mg/l). Further evaluation against selected mutants (TK- or PAAr) indicated a requirement for the expression of the virus-coded thymidine kinase (TK) for the antiviral activity of Ro 21-9875. Kinetic studies revealed non-competitive mixed inhibition of the viral enzyme by this compound. This suggests that it may have some intrinsic TK mediated activity though breakdown to its component parts is undoubtedly a significant contributing factor.

Cyclohexane triones, novel membrane-active antibacterial agents.

The cyclohexane triones are a novel group of synthetic antibacterial agents that are active against gram-positive bacteria, Haemophilus influenzae, and Mycobacterium smegmatis. In general, these compounds behaved in a manner similar to that of hexachlorophene, inhibiting the transport of low-molecular-weight hydrophilic substances into bacteria. Unlike cationic detergents, such as chlorhexidine, they did not cause disruption of the bacterial cytoplasmic membrane over a short time period. The most potent antibacterial cyclohexane trione studied had a reduced ability to inhibit solute transport in comparison with certain less active analogs. Cyclohexane triones may express more than a single type of antibacterial effect.

Nucleoside analogues as highly potent and selective inhibitors of herpes simplex virus thymidine kinase.

A series of carboxamide derivatives of 5'-amino-2',5'-dideoxy-5-ethyluridine has been prepared as inhibitors of HSV-TK (herpes simplex virus thymidine kinase). The most potent compounds were derived from xanthene, thioxanthene and dihydroanthracene carboxylic acids. The lead compounds show subnanomolar IC(50) values against HSV TKs.